Journal of Microbiology

Journal Article

Extended stability of cyclin D1 contributes to limited cell cycle arrest at G1-phase in BHK-21 cells with Japanese encephalitis virus persistent infection: Ji Young Kim , Soo Young Park , Hey Rhyoung Lyoo , Eung Seo Koo , Man Su Kim , Yong Seok Jeong; J. Microbiol. 2015;53(1):77-83. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4661-z

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Abstract: There is increasing evidence that many RNA viruses manipulate cell cycle control to achieve favorable cellular environments for their efficient replication during infection. Although virus-induced G0/G1 arrest often delays early apoptosis temporarily, a prolonged replication of the infected virus leads host cells to eventual death. In contrast, most mammalian cells with RNA virus persistent infection often escape cytolysis in the presence of productive viral replication. In this study, we demonstrated that the extended endurance of cyclin D1 was clearly associated with the suppression of glycogen synthase kinase-3β (GSK-3β) expression in BHK-21 cells that are persistently infected with Japanese encephalitis virus (JEV). The G0/G1 arrest of these cells turned much loose compared to the normal BHK-21 cells with JEV acute infection. After cycloheximide treatment, cyclin D1 in the persistently infected cells lasted several hours longer than those in acutely infected cells. Furthermore, both p21Cip1 and p27Kip1, positive regulators for cyclin D1 accumulation in the nucleus, were suppressed in their expression, which contrasts with those in JEV acute infection. Inhibition of the GSK-3β by lithium chloride treatment rescued a significant number of cells from cytolysis in JEV acute infection, which coincided with the levels of cyclin D1 that escaped from proteolysis. Therefore, the limitation of G1/S arrest in the BHK-21 cells with JEV persistent infection is associated with the suppression of GSK-3β expression, resulting in the extended duration of cyclin D1.

Validation Study

Comparison of JEV Neutralization Assay Using Pseudotyped JEV with the Conventional Plaque-Reduction Neutralization Test: Hee-Jung Lee , Kyung-Il Min , Ki Hoon Park , Hyo Jung Choi , Min-Kyoung Kim , Chi-Young Ahn , Young-Jin Hong , Young Bong Kim; J. Microbiol. 2014;52(5):435-440. Published online March 7, 2014; DOI: https://doi.org/10.1007/s12275-014-3529-y

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18 Citations

Abstract: We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV- PV). JEV-PV-based neutralization assay offers several advan-tages compared with the current standard plaque-reduc-tion neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque re-duction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.

Research Support, Non-U.S. Gov't

NOTE] Biological and Genetic Properties of SA14-14-2, a Live-Attenuated Japanese Encephalitis Vaccine That Is Currently Available for Humans: Byung-Hak Song , Gil-Nam Yun , Jin-Kyoung Kim , Sang-Im Yun , Young-Min Lee; J. Microbiol. 2012;50(4):698-706. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2336-6

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28 Citations

Abstract: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a major cause of acute encephalitis, a disease of significance for global public health. In the absence of antiviral therapy to treat JEV infection, vaccination is the most effective method of preventing the disease. In JE-endemic areas, the most widely used vaccine to date is SA14-14-2, a live-attenuated virus derived from its virulent parent SA14. In this study, we describe the biological properties of SA14-14-2, both in vitro and in vivo, and report the genetic characteristics of its genomic RNA. In BHK-21 (hamster kidney) cells, SA14-14-2 displayed a slight delay in plaque formation and growth kinetics when compared to a virulent JEV strain, CNU/LP2, with no decrease in maximum virus production. The delay in viral growth was also observed in two other cell lines, SH-SY5Y (human neuroblastoma) and C6/36 (mosquito larva), which are potentially relevant to JEV pathogenesis and transmission. In 3-week-old ICR mice, SA14-14-2 did not cause any symptoms or death after either intracerebral or peripheral inoculation with a maximum dose of up to 1.5×103 plaqueforming units (PFU) per mouse. The SA14-14-2 genome consisted of 10977 nucleotides, one nucleotide longer than all the previously reported genomes of SA14-14-2, SA14 and two other SA14-derived attenuated viruses. This difference was due to an insertion of one G nucleotide at position 10701 in the 3' noncoding region. Also, we noted a significant number of nucleotide and/or amino acid substitutions throughout the genome of SA14-14-2, except for the prM protein-coding region, that differed from SA14 and/or the other two attenuated viruses. Our results, together with others’, provide a foundation not only for the study of JEV virulence but also for the development of new and improved vaccines for JEV.

Characterization of an Attenuated Japanese Encephalitis Virus Adapted to African Green Nomkey Kidney Cells, Vero: Chung, Yong Ju , Hong, Sun Pyo , Moon, Sang Beom , Shin, Young Cheol , Kim, Soo Ok; J. Microbiol. 1998;36(3):189-195.

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Abstract: Live attenuated Japanese encephalitis (JE) virus SA14-14-2 produced in primary dog kidney cells (PDK) was adapted to African green monkey kidney cells, Vero. In an effort to gain insight into the molecular basis of the biological characteristics of the isolated SA14-14-2 (Vero) strain, the 1,500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and dompared with the sequences of two other attenuated JE virus strains, SA14-14-2 (PHK) and SA14-14-2 (PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) of the SA14-14-2 (Vero) E gene was found to be identical to those of strains SA14-14-2 (PHK) and SA14-14-2 (PDK), while the N-terminal region (a.a. 1-279) showed sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a.138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA14-14-2 (Vero). Animal testing showed that SA14-14-2 (Vero) has a neurovirulence phenotype similar to that of the parent SA14-14-2 (PDK) strain in suckling mice. The SA14-14-2 (Vero) grew very efficiently in Vero cells enough to support vaccine production. The growth characteristics of SA14-14-2 (Vero) in Vero cell and conservation of attenuation determinant of neurovirulence support that SA14-14-2 (Vero) could be developed as a new vaccine strain for human use.

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